Interferons (IFNs) were discovered in 1957 by Isaacs and Lindenmann, and were named for their ability to interfere with viral proliferation. Interferons can also combat bacterial and parasitic infections, inhibit cell division, inhibit spontaneous apoptosis, and promote or impede the differentiation of cells. Based on their receptor specificity, two types of interferons are recognized: Type I and Type II. The Type I interferons are a family of monomeric proteins including IFNα and IFNω that are products of leukocytes, IFNβ that is produced by fibroblasts, and IFNτ that has been described only in ungulate species.
The only known type II interferon the dimeric IFNγ, which is produced exclusively by lymphocytes. The interferon alpha family is composed of 13 intronless fully translated genes (excluding pseudogenes). Each member includes mature proteins of 165 or 166 amino acid residues, with two conserved disulfide bonds: Cys1-Cys98 and Cys29-Cys138. A high level of sequence homology (80%) is displayed among the various interferon alpha sub-types, and about 35% homology exists between these sub-types and the IFNβ. In spite of the high homology of the different sub-types, their biological activities, among them antiproliferative, antiviral, and immunomodulation, differ notably.
The structure of several type I interferons have been solved including murine IFNβ (1IFA, 1RMI), human IFNβ (1AU1), human IFNα2 (1RH2, 1ITF), and ovine IFNτ (1B5L). Structurally, interferons are members of the α-helical cytokine family. All the type I interferons signal through a common receptor complex composed of IFNAR1 and IFNAR2. The major ligand-binding component of the type I interferon receptor is IFNAR2, with a binding affinity of ˜10 nM for IFNα2. The structure of the extracellular part of IFNAR2 consists of two immunoglobulin-like domains, with the binding site of IFNα2 being located at the N-terminal domain and the connecting loop.
Mature IFNAR1 is a 530 amino acid protein, with a transmembrane segment composed of 21 residues, and a cytoplasmatic domain of 100 residues. The structure of the extracellular part of IFNAR1 is unknown, but from the sequence one can deduce that it is composed of four immunoglobulin-like domains. Binding of IFNα2 to IFNAR1 is weak, with the affinity measured on an artificial membrane being 1.5-5 μM. Bovine IFNAR1 (BoIFNAR1) and the human IFNAR1 (HuIFNAR1), are 68% identical. The sub-domains 2 and 3 of HuIFNAR1 have been shown to play a critical role in binding to IFNα2. By exchanging these sub-domains with homologous BoIFNAR1, the affinity was increased substantially. The soluble BoIFNAR1 can bind human interferons with a 10 nM affinity, which is 500-fold greater than that of HuIFNAR1. Murine cells expressing IFNAR2 bind IFNα2 with an affinity of 8 nM, whereas cells expressing only IFNAR1 exhibit no ligand binding. Upon co-expression of IFNAR1 and IFNAR2, a ten-fold increase in the affinity for IFNα2 was observed. Using in-vitro studies on artificial membranes it was shown that the magnitude of IFNAR1-induced increase in binding affinity of the ternary complex relates to the relative surface concentration of this receptor. The location of the IFNAR1 binding site on interferon was mapped on IFNβ to be located on the B, C, and D helices and the DE loop while Ala scan mutagenesis of IFNα2 have suggested that the IFNAR1 binding site is restricted to helices B and C.
A number of studies have suggested that the formation of the ternary complex occurs in a sequential mode, beginning with interferon binding IFNAR2 to form an intermediate complex, and followed by the recruitment of IFNAR1. The IFNAR1-IFNβ-IFNAR2 complex has been shown to have a 1:1:1 stoichiometry. The IFNAR1 receptor is an essential component of the interferon receptor complex, with an IFNAR1 null mutation, or the addition of neutralizing Ab against this receptor resulting in a complete lack of the antiviral and antiproliferative responses to IFNα and IFNβ. Association of IFNAR1 and IFNAR2, stimulates the activation of the constitutively-associated intracellular kinases Jak1 and Tyk2, leading to a tyrosine phosphorylation cascade that results in the dimerization of the phosphorylated signal transducers and activators of transcription (STATs), and transport into the nucleus, where they bind to specific DNA sequences and stimulate transcription of hundreds of responsive genes.
The question remains open on how very similar IFNs induce differential activities on the same cell type. It has been suggested that the biological activities of different IFNα sub-types correlate with their respective binding affinities and the cell type used.
The vertebrate type I interferons are recognized by a single shared receptor, composed of two transmembrane proteins (IFNAR1 and IFNAR2), present their activity through their associated Jak kinases with the Stat transcription factors as their main targets (Brierley, M. M. & Fish, E. N. 2002. J Interferon Cytokine Res. 22, 835-845). Typically recognized by the IgG-like folds of their extracellular domains (hCR domains), IFNAR2 and IFNAR1 are regarded respectively as binding proteins and accessory transducing factors—i.e., the alpha and beta chains of heteromeric receptors. A difference in ligand dissociation constants of the two chains is implicit in the definition. Nevertheless, both contribute to the creation of high affinity binding sites. The combination of a “common” beta chain with different recognition chains is a feature of heteromeric receptors that respond differentially to different ligands. The ability to interact with different alpha-chains establishes potential network connections for differential receptor expression (Kotenko, S. V. & Langer, J. A. 2004. Int Immunopharmacol 4, 593-608). When, like IFNAR1, they possess the capacity of interacting with elements of different signaling pathways, they may establish connections for differential gene expression (Platanias, L. C. & Fish, E. N. 1999. Experimental Hematology 27, 1583-1592).
The human IFNs number 12 distinct non-allelic alpha proteins, one beta and one omega. As expected from a family with marked sequence homology, a shared 3D core structure and a shared receptor, the activities of the Type I IFNs overlap. Nevertheless they can be recognized and even classified by their amino acid differences and numerous instances of relative differences in activity have been noted. The emerging picture is that functional differences appear only in specific physiological contexts. In addition of their local action in conferring antiviral protection on almost any cell, they are linked to the development of second line antiviral defenses. It was noted that a possible difference between the IFNs might be their potential to bind tightly to IFNAR1 (Roisman et al., 2005. J Mol. Biol. 353, 271-281).
The differential activities of Type I IFNs have been a subject of intense investigations over many years. Particularly it was noted that IFNβ has an additional repertoire of activities over IFNα. Detailed analysis of the differences between these two IFNs has shown that IFNβ has a general higher activity in transcription activation of IFN responsive genes, and is active at reduced IFN levels. Binding studies suggested that affinity towards the accessory subunit IFNAR1 is the key differences between IFNα2 and IFNβ (Jaitin, 2006. Mol Cell Biol. 26, 1888-1897).
IFNα2 is known to have anti-cancer effects. However, this treatment is not always effective and sometimes results in intolerable side effects related to the dosage and duration of therapy. WO 97/12630 discloses treating cancer patients with temozolomide in combination with IFNα2. WO 01/54678 discloses treating cancer patients with temozolomide and pegylated interferon.
Hepatitis C virus (HCV) infection is the most common chronic blood borne infection in the United States. Although the numbers of new infections have declined, the burden of chronic infection is substantial; the Center for Disease Control estimates 3.9 million (1.8%) infected persons in the United States. Chronic liver disease is the tenth leading cause of death among adults in the United States, and accounts for approximately 25,000 deaths annually, or approximately 1% of all deaths. Studies indicate that 40% of chronic liver disease is HCV-related, resulting in an estimated 8,000-10,000 deaths each year. HCV-associated end-stage liver disease is the most frequent indication for liver transplantation among adults.
Antiviral therapy of chronic hepatitis C has evolved rapidly over the last decade, with significant improvements seen in the efficacy of treatment. Nevertheless, even with combination therapy using pegylated IFN-α plus ribavirin, 40% to 50% of patients fail therapy, i.e., are non-responders or relapsers. These patients currently have no effective therapeutic alternative. In particular, patients who have advanced fibrosis or cirrhosis on liver biopsy are at significant risk of developing complications of advanced liver disease, including ascites, jaundice, variceal bleeding, encephalopathy, and progressive liver failure, as well as a markedly increased risk of hepatocellular carcinoma.
Multiple sclerosis (MS) is a chronic, neurological, autoimmune, demyelinating disease. MS can cause blurred vision, unilateral vision loss (optic neuritis), loss of balance, poor coordination, slurred speech, tremors, numbness, extreme fatigue, changes in intellectual function (such as memory and concentration), muscular weakness, paresthesias, and blindness. Many subjects develop chronic progressive disabilities, but long periods of clinical stability may interrupt periods of deterioration. Neurological deficits may be permanent or evanescent. The pathology of MS is characterized by an abnormal immune response directed against the central nervous system. In particular, T-lymphocytes are activated against the myelin sheath of the central nervous system causing demyelination. In the demyelination process, myelin is destroyed and replaced by scars of hardened “sclerotic” tissue which is known as plaque. These lesions appear in scattered locations throughout the brain, optic nerve, and spinal cord. The two types of interferon-beta that are approved in the United States for use in treating MS are interferon-beta 1a and interferon-beta 1b.
Type I diabetes, also known as autoimmune diabetes or insulin-dependent diabetes mellitus (IDDM), is an autoimmune disease characterized by the selective destruction of pancreaticss cells by autoreactive T lymphocytes (Bach, 1994, Endocr. Rev. 15:516-542). The pathology of IDDM is very complex involving an interaction between an epigenetic event (possibly a viral infection), the pancreatic islet cells and the immune system in a genetically susceptible host. A number of cytokines, including IFN-α and IFN-γ, have been implicated in the pathogenesis of IDDM in humans and in animal models of the disease (Campbell et al., 1991, J. Clin. Invest. 87:739-742). It appears that local expression of IFN-α by pancreatic islet cells in response to potential diabetogenic stimuli such as viruses may trigger the insulitic process.
WO9304699 discloses a method for treatment of insulin dependent diabetes mellitus comprising administering an IFN-α antagonist.
Based on the increased level of IFN-α expression in patients with systemic lupus erythematosus (SLE), IFN-α has also been implicated in the pathogenesis of SLE (Ytterberg and Schnitzer, 1982, Arthritis Rheum. 25: 401-406). WO02066649 discloses anti-IFN-α specific antibodies for the treatment of insulin-dependent diabetes mellitus (IDDM) and systemic lupus elythematosus (SLE).
US Patent Application Publication No. 20040230040 discloses cysteine variants of α interferon-2. US Patent Application Publication No. 20040002474 discloses α interferon homologues having antiproliferative activity in a human Daudi cell line-based assay.
U.S. Pat. No. 4,588,585 discloses mutated IFN-β-1b in which Cys17 is changed to Ser17 via a T to A transition in the first base of codon 17, which prevents incorrect disulfide bond formation. WO2005016371 discloses a pharmaceutical composition comprising an improved recombinant human IFN-β-1b variant with a greater specific activity.
The available treatments for cancer, infectious diseases, multiple sclerosis, and autoimmune disorders associated with increased expression of IFNα2 are expensive, effective only in a certain percentage of patients and adverse side effects are not uncommon. There remains an unmet medical need for suitable therapeutic methods that are safe, reliable, efficacious, and cost effective.